Identification of Shell Colour Pigments in Marine Snails Clanculus pharaonius and C. margaritarius (Trochoidea; Gastropoda).

Colour and pattern are key traits with important roles in camouflage, warning and attraction. Ideally, in order to begin to understand the evolution and ecology of colour in nature, it is important to identify and, where possible, fully characterise pigments using biochemical methods. The phylum Mollusca includes some of the most beautiful exemplars of biological pigmentation, with the vivid colours of sea shells particularly prized by collectors and scientists alike. Biochemical studies of molluscan shell colour were fairly common in the last century, but few of these studies have been confirmed using modern methods and very few shell pigments have been fully characterised. Here, we use modern chemical and multi-modal spectroscopic techniques to identify two porphyrin pigments and eumelanin in the shell of marine snails Clanculus pharaonius and C margaritarius. The same porphyrins were also identified in coloured foot tissue of both species. We use high performance liquid chromatography (HPLC) to show definitively that these porphyrins are uroporphyrin I and uroporphyrin III. Evidence from confocal microscopy analyses shows that the distribution of porphyrin pigments corresponds to the striking pink-red of C. pharaonius shells, as well as pink-red dots and lines on the early whorls of C. margaritarius and yellow-brown colour of later whorls. Additional HPLC results suggest that eumelanin is likely responsible for black spots. We refer to the two differently coloured porphyrin pigments as trochopuniceus (pink-red) and trochoxouthos (yellow-brown) in order to distinguish between them. Trochopuniceus and trochoxouthos were not found in the shell of a third species of the same superfamily, Calliostoma zizyphinum, despite its superficially similar colouration, suggesting that this species has different shell pigments. These findings have important implications for the study of colour and pattern in molluscs specifically, but in other taxa more generally, since this study shows that homology of visible colour cannot be assumed without identification of pigments.

The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

Yeast metabolic engineering for hemicellulosic ethanol production.

Efficient fermentation of hemicellulosic sugars is critical for the bioconversion of lignocellulosics to ethanol. Efficient sugar uptake through the heterologous expression of yeast and fungal xylose/glucose transporters can improve fermentation if other metabolic steps are not rate limiting. Rectification of cofactor imbalances through heterologous expression of fungal xylose isomerase or modification of cofactor requirements in the yeast oxidoreductase pathway can reduce xylitol production while increasing ethanol yields, but these changes often occur at the expense of xylose utilization rates. Genetic engineering and evolutionary adaptation to increase glycolytic flux coupled with transcriptomic and proteomic studies have identified targets for further modification, as have genomic and metabolic engineering studies in native xylose fermenting yeasts.

A rapid and reliable method for Pb isotopic analysis of peat and lichens by laser ablation-quadrupole-inductively coupled plasma-mass spectrometry for biomonitoring and sample screening.

An analytical protocol for rapid and reliable laser ablation-quadrupole (LA-Q)- and multi-collector (MC-) inductively coupled plasma-mass spectrometry (ICP-MS) analysis of Pb isotope ratios ((207)Pb/(206)Pb and (208)Pb/(206)Pb) in peats and lichens is developed. This technique is applicable to source tracing atmospheric Pb deposition in biomonitoring studies and sample screening. Reference materials and environmental samples were dry ashed and pressed into pellets for introduction by laser ablation. No binder was used to reduce contamination. LA-MC-ICP-MS internal and external precisions were <1.1% and <0.3%, respectively, on both (207)Pb/(206)Pb and (208)Pb/(206)Pb ratios. LA-Q-ICP-MS internal precisions on (207)Pb/(206)Pb and (208)Pb/(206)Pb ratios were lower with values for the different sample sets <14.3% while external precisions were <2.9%. The level of external precision acquired in this study is high enough to distinguish between most modern Pb sources. LA-MC-ICP-MS measurements differed from thermal ionisation mass spectrometry (TIMS) values by 1% or less while the accuracy obtained using LA-Q-ICP-MS compared to solution MC-ICP-MS was 3.1% or better using a run bracketing (RB) mass bias correction method. Sample heterogeneity and detector switching when measuring (208)Pb by Q-ICP-MS are identified as sources of reduced analytical performance.

Biogeochemical signatures in the lichen Hypogymnia physodes in the mid Urals.

Multi-element content and uranium (U) isotopes were investigated in the lichen Hypogymnia physodes (native and transplants) sampled across a 60-km transect, centred on Karabash smelter town, from Turgoyak Lake (SW) to Kyshtym (NE) to investigate the origin of U. Kyshtym was the site of a major nuclear accident in 1957. (234)U/(238)U activity ratios in native thalli sampled during July 2001 were within the natural isotopic ratio in minerals. Uranium/thorium (U/Th) ratios were higher in native thalli towards the NE (average 0.73) than those in the SW (average 0.57). Element signatures in native thalli and transplants suggest U was derived from fossil fuel combustion from Karabash and sources lying further to the east. Systematic and significant U enrichment indicative of a nuclear fuel cycle source was not detected in any sample. Element signatures in epiphytic lichen transplants and native thalli provide a powerful method to evaluate U deposition.

Metabolic engineering for improved fermentation of pentoses by yeasts.

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g(-1) sugar consumed, so commercialization seems feasible for some applications.

Bacteria engineered for fuel ethanol production: current status.

The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.

Which factors are responsible for the changing lichen floras of London?

SO(2) is no longer the principal factor influencing the vitality and composition of lichen assemblages in London. We provide direct evidence for an impact on lichen growth during episodic high exhaust emissions coupled with unusual climatic conditions. This suggests a combination of particles and nitrogen plays a major role in influencing lichen growth. Nitrogen from traffic emissions may be at least as important as agriculture in influencing the composition of lichen assemblages.

Developmental and morphological regulation of clathrin-mediated endocytosis in Trypanosoma brucei.

Essentially all macromolecular communication between Trypanosoma brucei and its host is confined to vesicular trafficking events occurring at or around the flagellar pocket. The vertebrate stage bloodstream form trypomastigote exhibits an extremely high rate of endocytosis required for nutrient uptake and probably also evasion of the host immune system. However, the rate of endocytosis is very low in the procyclic vector parasite, indicating that endocytosis is subject to a marked level of developmental regulation. Previous ultrastructural studies and crude biochemical fractionations have indicated the presence of coated pits and vesicles that are analogous to clathrin coats in the bloodstream form, but not in the procyclic. However, a definitive description of the components of this coat and its molecular function in T. brucei has remained elusive. We describe the molecular cloning and initial characterisation of components of the T. brucei endocytic coats: clathrin heavy chain (TbCLH) and a beta-adaptin (TbAPbeta1). TbCLH is markedly upregulated in the bloodstream form compared with the procyclic, whereas TbAPbeta1 is subject to more limited developmental regulation. We generated antisera against both proteins and show that the clathrin coat is tightly associated with the flagellar pocket in both major life stages. However, in bloodstream parasites TbCLH is also extensively distributed throughout the posterior end of the cell on numerous large vesicular and tubular structures. By cryo-immuno EM, clathrin is localised to collecting tubules at the flagellar pocket and is also associated with the trans-Golgi network. These EM data confirm that the electron dense coats reported on trypanosome vesicles and tubules contain clathrin. The TbAPbeta1 exhibits an atypical distribution relative to previously characterised adaptins, associating not only with the trans-Golgi but also with other tubular-vesicular elements. Localisation of TbAPbeta1 is also subject to developmental regulation. These data describe major endocytic coat proteins in T. brucei for the first time, and indicate stage-specific expression of the clathrin heavy chain. Modulation of clathrin expression is likely to be an important factor in the developmental regulation of endocytosis and recycling in the African trypanosome.

A developmentally regulated rab11 homologue in Trypanosoma brucei is involved in recycling processes.

Endocytosis in the parasitic protozoan Trypanosoma brucei, a deeply divergent eukaryote, is implicated as important in both general cellular function and virulence, and is strongly developmentally regulated. We report the characterisation of a previously undefined endosomal compartment in T. brucei based on identification of a new trypanosome gene (TbRAB11) homologous to Rabll/Ypt31. Northern and western analyses indicated that TbRAB11 expression was significantly upregulated in the bloodstream stage of the parasite, the first trypanosome Rab to be identified with a developmentally regulated expression profile. In procyclic form parasites TbRAB11 localised to a compartment positioned close to the basal body, similar to mammalian Rab11. By contrast, in bloodstream form parasites, TbRAB11-containing structures were more extensive and the TbRAB11 compartment extended towards the posterior face of the nucleus, was more elaborate and was not always adjacent to the basal body. Colocalisation studies by light and confocal microscopy demonstrated that TbRAB11 was located on a compartment that did not correspond to other established trypanosomal organelles or markers. Using concanavalin A internalisation and temperature block procedures, TbRAB11 was observed on endomembranes anterior to the flagellar pocket that are juxtaposed to the collecting tubules. TbRAB11 colocalised with the trypanosomal transferrin receptor and internalised anti-variant surface glycoprotein. Further, we show that the collecting tubules contain TbRAB5A, suggesting that they are the trypanosomatid early endosome. Hence, TbRAB11 is present on endosomal structures that contain recycling cargo molecules and is under developmental regulation, suggesting a role in stage-dependent endocytic processes.

A new astrophysical setting for chondrule formation.

Chondrules in the metal-rich meteorites Hammadah al Hamra 237 and QUE 94411 have recorded highly energetic thermal events that resulted in complete vaporization of a dusty region of the solar nebula (dust/gas ratio of about 10 to 50 times solar). These chondrules formed under oxidizing conditions before condensation of iron-nickel metal, at temperatures greater than or equal to 1500 K, and were isolated from the cooling gas before condensation of moderately volatile elements such as manganese, sodium, potassium, and sulfur. This astrophysical environment is fundamentally different from conventional models for chondrule formation by localized, brief, repetitive heating events that resulted in incomplete melting of solid precursors initially residing at ambient temperatures below approximately 650 K.

Chronic toxicity and oncogenicity studies of ingested 1, 3-dichloropropene in rats and mice.

Fischer 344 rats and B6C3F1 mice were administered 1, 3-dichloropropene (1,3-D) via their diets for up to 2 years, at dose levels of 0, 2.5, 12.5, or 25 mg 1,3-D/kg body wt/day for rats and 0, 2.5, 25, or 50 mg 1,3-D/kg body wt/day for mice. The test material was stabilized in the feed by microencapsulation in a starch/sucrose matrix (80/20%). Rats given 12.5 or 25 mg/kg/day, and mice given 25 or 50 mg/kg/day, had decreased body weights and body weight gains. There were no effects on survival or clinical pathology parameters for rats or mice. Histopathologic effects attributed to treatment in rats consisted of basal cell hyperplasia of the nonglandular mucosa of the stomach in males and females given 12.5 or 25 mg/kg/day for 12 and 24 months and an increased number of hepatocellular adenomas in males given 12.5 or 25 mg/kg/day and females given 25 mg/kg/day for 24 months. The increase in hepatocellular adenomas was statistically identified by pairwise comparison only in males given 25 mg/kg/day. An increased incidence of eosinophilic foci of altered cells in the liver was also noted in all treated groups of rats at 24 months. The latter observation, however, was considered of equivocal toxicological significance because of the common spontaneous occurrence of liver foci in aged Fischer 344 rats. The only histologic change attributed to treatment in mice was decreased size of hepatocytes in males given 50 mg/kg/day for 12 months. The decreased size of hepatocytes was consistent with decreased cytoplasmic glycogen content and corresponded to decreased liver weights. This effect was not present at 24 months. There was no oncogenic response observed in mice. The low-dose level of 2.5 mg/kg/day was interpreted as the no-observed-adverse-effect level (NOAEL) for systemic chronic toxicity of 1,3-D in the Fischer 344 rat. The no-observed-effect level (NOEL) for chronic systemic toxicity was 2.5 mg/kg/day in the B6C3F1 mouse.

Characterization and complementation of a Pichia stipitis mutant unable to grow on D-xylose or L-arabinose.

Pichia stipitis CBS 6054 will grow on D-xylose, D-arabinose, and L-arabinose. D-Xylose and L-arabinose are abundant in seed hulls of maize, and their utilization is important in processing grain residues. To elucidate the degradation pathway for L-arabinose, we obtained a mutant, FPL-MY30, that was unable to grow on D-xylose and L-arabinose but that could grow on D-arabinitol. Activity assays of oxidoreductase and pentulokinase enzymes involved in D-xylose, D-arabinose, and L-arabinose pathways indicated that FPL-MY30 is deficient in D-xylitol dehydrogenase (D-XDH), D- and L-arabinitol dehydrogenases, and D-ribitol dehydrogenase. Transforming FPL-MY30 with a gene for xylitol dehydrogenase (PsXYL2), which was cloned from CBS 6054 (GenBank AF127801), restored the D-XDH activity and the capacity for FPL-MY30 to grow on L-arabinose. This suggested that FPL-MY30 is critically deficient in XYL2 and that the D-xylose and L-arabinose metabolic pathways have xylitol as a common intermediate. The capacity for FPL-MY30 to grow on D-arabinitol could proceed through D-ribulose.

Ethanol and thermotolerance in the bioconversion of xylose by yeasts.

The mechanisms underlying ethanol and heat tolerance are complex. Many different genes are involved, and the exact basis is not fully understood. The integrity of cytoplasmic and mitochondrial membranes is critical to maintain proton gradients for metabolic energy and nutrient uptake. Heat and ethanol stress adversely affect membrane integrity. These factors are particularly detrimental to xylose-fermenting yeasts because they require oxygen for biosynthesis of essential cell membrane and nucleic acid constituents, and they depend on respiration for the generation of ATP. Physiological responses to ethanol and heat shock have been studied most extensively in S. cerevisiae. However, comparative biochemical studies with other organisms suggest that similar mechanisms will be important in xylose-fermenting yeasts. The composition of a cell's membrane lipids shifts with temperature, ethanol concentration, and stage of cultivation. Levels of unsaturated fatty acids and ergosterol increase in response to temperature and ethanol stress. Inositol is involved in phospholipid biosynthesis, and it can increase ethanol tolerance when provided as a supplement. Membrane integrity determines the cell's ability to maintain proton gradients for nutrient uptake. Plasma membrane ATPase generates the proton gradient, and the biochemical characteristics of this enzyme contribute to ethanol tolerance. Organisms with higher ethanol tolerance have ATPase activities with low pH optima and high affinity for ATP. Likewise, organisms with ATPase activities that resist ethanol inhibition also function better at high ethanol concentrations. ATPase consumes a significant fraction of the total cellular ATP, and under stress conditions when membrane gradients are compromised the activity of ATPase is regulated. In xylose-fermenting yeasts, the carbon source used for growth affects both ATPase activity and ethanol tolerance. Cells can adapt to heat and ethanol stress by synthesizing trehalose and heat-shock proteins, which stabilize and repair denatured proteins. The capacity of cells to produce trehalose and induce HSPs correlate with their thermotolerance. Both heat and ethanol increase the frequency of petite mutations and kill cells. This might be attributable to membrane effects, but it could also arise from oxidative damage. Cytoplasmic and mitochondrial superoxide dismutases can destroy oxidative radicals and thereby maintain cell viability. Improved knowledge of the mechanisms underlying ethanol and thermotolerance in S. cerevisiae should enable the genetic engineering of these traits in xylose-fermenting yeasts.

Genetic engineering for improved xylose fermentation by yeasts.

Xylose utilization is essential for the efficient conversion of lignocellulosic materials to fuels and chemicals. A few yeasts are known to ferment xylose directly to ethanol. However, the rates and yields need to be improved for commercialization. Xylose utilization is repressed by glucose which is usually present in lignocellulosic hydrolysates, so glucose regulation should be altered in order to maximize xylose conversion. Xylose utilization also requires low amounts of oxygen for optimal production. Respiration can reduce ethanol yields, so the role of oxygen must be better understood and respiration must be reduced in order to improve ethanol production. This paper reviews the central pathways for glucose and xylose metabolism, the principal respiratory pathways, the factors determining partitioning of pyruvate between respiration and fermentation, the known genetic mechanisms for glucose and oxygen regulation, and progress to date in improving xylose fermentations by yeasts.

Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production.

The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization to CYC1 from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1 showed 74% and 69% identity to ScCYC1 and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1 resulted in a mutant that uses the salicylhydroxamic acid (SHAM)-sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes c and a.a(3) both disappeared in the cyc1-Delta mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1-Delta mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1-Delta mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0.46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0.38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen-limiting conditions.

Bioconversion of secondary fiber fines to ethanol using counter-current enzymatic saccharification and co-fermentation.

This research examined several enzymatic and microbial process for the conversion of waste cellulosic fibers into ethanol. The first was a one-stage process in which pulp fines were contacted with commercial enzyme solutions. The second process used sequential, multistage saccharification. The third used sequential enzyme addition in a countercurrent mode. Experiments compared the results with various feedstocks, different commercial enzymes, supplementation with beta-glucosidase, and saccharification combined with fermentation. The highest saccharification (65%) from a 4% consistency pulp and the highest sugar concentration (5.4%) from an 8% consistency pulp were attained when 5 FPU/g plus 10 IU/g of beta-glucosidase were used. Sequential addition of enzyme to the pulp in small aliquots produced a higher overall sugar yield/U enzyme than the addition of the same total amount of enzyme in a single dose. In the saccharification and fermentation experiments, we produced 2.12% ethanol from a 5.4% sugar solution. This represents 78% of the theoretical maximum. This yield could probably be increased through optimization of the fermentation step. Even when little saccharification occurred, the enzyme facilitated separation of water, fiber, and ash, so cellulase treatment could be an effective means for dewatering pulp sludges.

Transcriptional control of ADH genes in the xylose-fermenting yeast Pichia stipitis.

We studied the expression of the genes encoding group I alcohol dehydrogenases (PsADH1 and PsADH2) in the xylose-fermenting yeast Pichia stipitis CBS 6054. The cells expressed PsADH1 approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of PsADH2 were not detectable under either aeration condition. We used a PsADH1::lacZ fusion to monitor PsADH1 expression and found that expression increased as oxygen decreased. The level of PsADH1 transcript was repressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of PsADH1, PsCYC1 expression was repressed. These results indicate that oxygen availability regulates PsADH1 expression and that regulation may be mediated by heme. The regulation of PsADH2 expression was also examined in other genetic backgrounds. Disruption of PsADH1 dramatically increased PsADH2 expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of PsADH2 is subject to feedback regulation under these conditions.

Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae.

Respiratory and fermentative pathways coexist to support growth and product formation in Pichia stipitis. This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions. Expression of Saccharomyces cerevisiae URA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present. ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth. Initial P. stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose. Cells produced even more ethanol faster following two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism. This is the first report of genetic engineering to enable anaerobic growth of a eukaryote.